The overall goal of this project is to further develop the strategy of gene therapy combined with radiotherapy for the treatment of human cancers. This project is a direct extension of specific aim 2 of the prior funding period of this project and is based on extensive progress and preliminary data generated during the last 5 years of support. The primary strategies to be developed involve "gene replacement", i.e. wild-type tumor suppressor genes will be transfected into tumor cells deficient in these genes. Certain genes of this type restore apoptosis propensity and/or restore normal cell cycle checkpoint control and would, therefore, be expected to have radiosensitizing abilities. Thus, the gone therapy vectors to be examined in this context will have dual effects in that they will have antitumor activity when used as single agents and, in addition, have radiosensitizing properties. A specific vector has already been generated that will be tested in detail: adenoviral-mediated mda7 (Ad-mda7). Specifically we propose to answer the following questions: 1) Does Ad-mda7 function to preferentially radiosensitize human tumor cells in vitro? The cells will be grown and treated in vitro and clonogenic survival will be the endpoint. 2) Does Ad-mda7 function to radiosensitize tumor microvascular endothelial cells? Here, we propose to test endothelial cells growing in vitro for radiosensitization by mda7 protein. Cells will be grown and treated in vitro and clonogenic survival will be the endpoint. 3) Does Ad-mda7 function to radiosensitize human xenograft tumorsin vivo? The human tumor cell lines shown to be radiosensitized in aim 1 above will be grown as xenograft tumors in nude mice and treated with Ad-mda7 and radiation in vivo. Tumor growth delay will be the endpoint. 4) What is the molecular mechanism by which Ad-mda7 radiosensitizes human tumor cells? Understanding the molecular basis for the radiosensitization imparted by this gene therapy strategy will enable us to design better gene therapy vectors in the future. Thus, under this aim, we propose to examine those specific molecular pathways of apoptosis, signal transduction and DNA repair known to be affected by this vector that may explain radiosensitization. Knowledge gained through this research will hopefully lead to the development of new, more-effective strategies for combining gene therapy and radiotherapy for the treatment of humar cancer.